Nitric Oxide (NO) has recently been shown to be a key bioregulatory molecule in a number of physiological processes. For example, NO plays a major role in the biological activity of endothelium derived relaxing factor (EDRF), abnormalities in which are associated with acute hypertension, diabetes, ischaemia and atherosclerosis. NO is also considered a retrograde messenger in the central nervous system, appears to be involved in the regulation of macrophage cytotoxic activity and platelet aggregation inhibition, and has been implicated in endotoxic shock and genetic mutations. In addition, a number of drugs and other xenobiotics are metabolized to produce NO as either the effector molecule or as a harmful metabolite. Many tissues in the body endogenously release NO in different amounts but the actual amount released are very difficult to quantify. In addition, many diseases, such as ischemia reperfusion injury, which include deamination-related genetic diseases like deamination of cytosine to thymine, cancer, and male impotence, have been suggested to be caused by defects in the production and/or regulation of NO. See e.g. Furchgott, R. F. et al., Nature 288:373-376 (1980); Palmer, R. M. et al., Nature 327:524-526 (1987); Furchgott, R. F., "Mechanism of Vasodilation", IV:401-414 (ed. Vanhoutte, P. M.) (Raven, N.Y.) (1988); Ignaro, L. J. et al., PNAS (USA) 84:9265-9269 (1987); Wel, E. P. et al., Cir. Res. 57:781-787 (1985); Piper, G. M. et al., J. Am. J. Physiol. 24:4825-4833 (1988); Vanbethuysen, K. M. et al., J. Clin. Invest. 79:265-274 (1987); Frelman, P. C. et al., Circ. Res. 58:783-789 (1986); and Schuman, E. M. et al., Science 254:1503 (1991).
The importance of the bioregulation effected by NO is further evidenced by the recent rash of pharmaceutical companies designing drugs around NO. It is hoped that drugs can be developed to control blood pressure, prevent atherosclerosis, treat migraine headaches and impotence, prevent deaths from septic shock, and help protect brain cells threatened by degenerative diseases and strokes.
Accordingly, the ability to specifically and quantitatively measure NO concentrations in solutions, particularly aqueous solutions of biological media, both in vitro and in vivo, and chemical media would be highly advantageous. The ability to measure the concentration of NO by a nondestructive method is an important requirement for further investigation of the mode of action of NO as a key bioregulatory molecule and for the development of therapeutic applications of NO-releasing compounds.
Several different methods have been employed in the past to measure NO concentration in aqueous solution. An automated system analyzes nitrate by reduction with a high-pressure cadmium column to determine amounts of nitrate and/or nitrite in urine, saliva, deproteinized plasma, gastric juice, and milk samples (Green et al., Analytical Biochemistry 126: 131-138. 1982.). The lower limit of detection of the method is said to be 1.0 nmol NO3.sub.3.sup.- or NO.sub.2.sup.- /ml. The system reportedly allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in biological fluids. Most samples may be prepared by simple dilution with distilled water, and 30 samples reportedly may be analyzed in an hour. The disadvantage of such a technique in measuring NO is that it does so indirectly, by measuring NO byproducts, which also can be generated from other sources. Accordingly, such a method is not very accurate in determining NO concentration.
Another method quantitatively analyzes nitrite, an oxidation product of NO, in human plasma to determine NO concentration (Wennmalm et al., Analyt. Biochem. 187:359-363. 1990). Dithionite is used to treat the samples of human plasma to convert nitrite to nitric oxide, with the treated samples being passed over bovine homoglobin columns. NO is allowed to bind the hemoglobin in columns of bovine hemoglobin covalently bound to agarose. An excess of dithionite is used to ensure that the hemoglobin is reduced to a ferrous, nonoxygenated state. The NO bound to the hemoglobin forms a complex on the column, and the column is then subjected to electron paramagnetic resonance spectrometry, i.e., the column is subjected to a magnetic field and microwave radiation to obtain a characteristic electron paramagnetic resonance spectrum. This method suffers from the same disadvantages as the previously described method. NO concentration is determined indirectly, through the measurement of nitrite. Also, the NO is modified by binding to hemoglobin covalently bound to agarose.
Other methods employed to quantitate NO include chemiluminescence, mass spectrometry (Bazylinski et al., Inorg. Chem. 24: 4285-4288. 1985), and ultraviolet-visible light spectral changes. In one procedure utilizing chemiluminescence, NO has been quantified by chemiluminescence resulting from the product of NO and ozone (Palmer et al., Nature 327: 524-526. 1987); Maragos et al., J. Med. Chem. 34: 3242-3247. 1991). This method also involves modification of NO, in this case by reaction with ozone. In a procedure employing ultraviolet and visible light, spectral changes have been monitored for the conversion of oxyhemoglobin to methemoglobin by NO as an indication of NO concentration (Haussman et al.). NO is modified in this method by reaction with oxyhemoglobin. Accordingly, neither one of these methods enables the measurement of NO directly.
Solution methods have been also used to measure NO but seem to lack specificity for NO or reliable quantitation. The use of 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS) as a spin trap in an electron spin resonance technique to detect NO in a biological system has been reported (Arroyo et al., Biophys. Res. Comm. 170: 1177-1183. 1990). This method, consequently, involves reaction of NO with modified spin traps. Subsequently, it was demonstrated that the obtained signal may result from simple oxidation of the spin trap, which raises the issue of how specific the spin trap is for NO (Wink et al., Radiat. Phys. Chem. 38: 467-472. 1991). The use of Fe.sup.2+ (dithiolate) to trap NO as the nitrosyl also has been used in a spin resonance technique (Mulsch et al., FEBS Letter 294: 252-256. 1991.); however, this technique is not suitable for quantitation due to a lack of biological stability, i.e., the resulting nitrosyl has a half-life of only about 30 seconds in biological systems. Further, it is evident that this method involves the modification of NO by formation of a complex with iron. The iron complex is metabolized, i.e., destroyed, during the process. Also, this method suffers from nitrite interference.
More recently, a modified oxygen electrode has been used to detect NO (Shibuki et al., Neuroscience Res. 9: 69-76. 1990.; Nature 349: 326-328. 1991.). The electrochemical microprobe was developed to detect the release of NO in brain tissue. The output current of the probe was found to correlate linearly with the concentration of NO at the tip. The sensitivity of the probe was reportedly between 3.5 and 106 pA/.mu.M change in NO concentration. However, the validity of this technique has been questioned due to the small current that has been observed (&lt;0.5 pA) and the lack of use of standards at submicromolor concentrations of NO. In addition, the technique measures NO by its oxidation to nitrite, and those who developed the modified oxygen electrode claim that NO is spontaneously released from sodium nitroprusside and that the release is accurately measured by the electrode. This contradicts what has been shown previously by others, i.e., that sodium nitroprusside does not spontaneously release NO in buffer (Kruszyna et al., Toxicol. Appl. Pharmacol. 91: 429-438. 1987.; Wilcox et al., Chem. Res. Toxicol. 3: 71-76. 1990.), which raises the issue of specificity of this method. Further, this electrode has a relatively large diameter (0.25 mm), a slow response time and a narrow concentration range (1-3 .mu.M). Although this method is advantageous in that it discriminates against the NO.sub.2.sup.- produced in the outer solution of the electrode, it is not selective for NO in the presence of any NO.sub.2.sup.- produced in the electrode inner solution.
Although the above-described methods can be used to measure NO in biological or chemical media, they are not sufficiently sensitive nor specific to provide a direct and accurate quantitative measurement of NO, particularly at low NO concentrations. Furthermore, none of the methods or sensors employed to date can rapidly and selectively measure NO release by the cell in situ in the presence of oxygen and/or NO.sub.2.sup.-. Development of this methodology is crucial in order to evaluate endogenous NO release, distribution and reactivity on molecular level in biological systems.
Thus, there exists a need for a sensitive and selective sensor for direct quantitive measurements of NO. An optimal sensor for monitoring NO release should be sturdy and capable of sufficient miniaturization for in situ measurement in a single cell. The sensor should also be sensitive enough to produce an adequate signal to be observable at the low levels of NO secreted in biological environments. Due to the variation in the amount of NO secreted by different types of cells (e.g. from nanomoles/10.sup.6 cells in macrophages to picomoles in endothelial cells), the signal produced by the sensor should also change linearly over a wide range of concentrations. See Marietta, M. A., Trends Biochem. Sci. 14:488-492 (1989). The short half-life of NO in biological systems, on the order of about 3-50 seconds, also mandates a fast response time. See Moncada, S. et al., Pharmacol. Rev. 43:109-142 (1991). The NO sensor and method of the present invention exhibit these desirable characteristics.